PROJECT SUMMARY Malaria remains a major global infectious disease, largely affecting people living in resource poor environments, and is one of the most important causes of childhood mortality. Drug-resistance is constantly undermining the usefulness of antimalarial regimens. There is an urgent need for the development of new therapeutic strategies. Plasmodium falciparum parasites utilize multiple ligand-receptor interactions for the invasion of human red blood cells, many of which are redundant. Different parasite strains rely on alternative ligand-interactions known as invasion pathways, for invasion, presumably for immune evasion and to invade polymorphic red blood cells. We have recently developed methods for the in vitro genetic analysis of red blood cell genes using hematopoietic stem cells. We have functionally characterized two red blood cell proteins, BSG and CD55, and have shown that they are essential for invasion by all P. falciparum strains, suggesting that they are strain- transcendent, prioritizing their interactions as targets for therapeutic development over strain-specific interactions. In forward genetic knockdown screens that assess all of membrane proteins found in the RBC proteome, we have identified a short list of high priority red blood cell determinants that we hypothesize are involved in strain-transcendent essential interactions between the host red blood cell and malaria parasite. In this proposal we will validate these genes as red blood cell determinants of strain- transcendent or strain- specific invasion. For this we will also leverage our development of CRISPR/Cas9-mediated gene editing in an erythroid cell-line that supports P. falciparum invasion, and facilitates the knockout of RBC proteins. The precise step of P. falciparum invasion that is mediated by each red blood cell determinant will be identified. We will use the RBC mutants that we generate to identify specific parasite ligands using both candidate and unbiased screening approaches. Together, these studies will serve to shift the paradigm from a focus on alternative redundant invasion pathways to the functional analysis of essential strain-transcendent host- parasite interactions. In the long-term we hope that our studies will provide a functional understanding of critical ligand-receptor interactions for P. falciparum invasion of erythrocytes to inform vaccine development and the design of host-targeted therapeutics.